The TCR:CD3 complex is one of the most important receptors in the immune system. The central theme and long-term objective of this project is to 'determine how TCR:CD3 complexity mediates the multitude of downstream functional events orchestrated by T cells.' The TCR:CD3 complex is unique in having ten ITAMs, but the physiological significance of this is unclear. In addition to the 25 publications contributed by this project, we have developed a powerful approach to dissect the contribution of CD3 ITAMs in mediating T cell development and function, which combines retroviral-mediated stem cell gene transfer and 2A peptide-linked multicistronic retroviral vectors. This allowed us to generate 25 groups of 'retrogenic' mice expressing different combinations of wild type and ITAM mutant TCR:CD3 complexes. Our analysis revealed dramatic and surprising differences in the TCR signal required for induction of proliferation, that correlated with ITAM number, versus cytokine production, which was unaffected by ITAM number. The molecular and signaling mechanisms that account for this striking disconnection are unknown. A key challenge now is to understand how the TCR utilizes CD3 ITAM complexity (number, flavor and location) to mediate the multitude of events that it initiates. In this project, we will use our unique CD3 retrogenic approach, advanced microscopy [time- lapse spinning disk confocal microscopy, with wide-field deconvolution, and total internal reflected fluorescent microscopy (TIRFM) with T cell:APC couples and anti-TCRb/ICAM or MHC class II/ICAM supported lipid bilayers], multiplexed, and conventional biochemical and functional assays. AIM 1: Mechanistic basis for the differential CD3 ITAM requirement for CD4+ T cell proliferation versus cytokine production. Here we will determine why a different number of functional CD3 ITAMs is required for mediating proliferation versus cytokine production in two sub-Aims: (A) How does CD3 ITAM number affect IS assembly, kinetics and stability? (B) How does CD3 ITAM number affect the initiation and transmission of TCR signaling? AIM 2: The relative contribution of CD3 ITAM flavor and location in mediating TCR signaling and T cell function. Our preliminary studies suggest that CD3 ITAM diversity is important as mice expressing CD3 complexes that contain a single ITAM 'flavor' (e.g. all ITAMs mutated to the same ITAM sequence) exhibit profound defects in T cell function and surprisingly divergent cytokine responses. The location of individual ITAMs within the three dimensional organization of the TCR:CD3 complex may also be important. These issues will be addressed in three sub-Aims: (A) What is the consequence of limiting CD3 ITAM diversity on T cell function? (B) What is the consequence of limiting CD3 ITAM diversity on TCR signaling? (C) How important is CD3 ITAM location within the TCR complex in mediating signaling and T cell function? PUBLIC HEALTH RELEVANCE: The T cell receptor (TCR):CD3 complex is one of the most important, complicated and intricate receptor complexes of the immune system, through which almost all immune responses are transmitted. Defects in TCR function can lead to autoimmunity or chronic inflammatory diseases. However, there is still much to be learnt about how TCR signaling is initiated, transmitted, disseminated and regulated. This application will focus on gaining a greater understanding of TCR function which may lead to novel therapeutic interventions.